Rhodopsin kinase structure: different nucleotide-binding states and implications for mechanism of activation of a G protein coupled receptor kinase

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Title: Rhodopsin kinase structure: different nucleotide-binding states and implications for mechanism of activation of a G protein coupled receptor kinase
Author: Singh, Puja, 1979-
Abstract: G protein coupled receptor (GPCR ) kinases (GRKs ) phosphorylate activated heptahelical receptors , leading to their uncoupling from G proteins and downregulation . The desensitization of GPCRs is critical to render cells responsive to further stimuli and if not regulated can result in many pathophysiological processes including heart abnormalities and hypertension . How GRKs recognize and are activated by GPCRs are not known , in part because the critical N -terminus and the kinase C -terminal extension were not resolved in GRK2 and GRK6 structures . The long -term goal of this project was to address this question by structural analysis of rhodopsin kinase (also known as GRK1 ) , which represents a model system for studying phosphorylation -dependent desensitization of activated GPCRs . Herein we report structures of GRK1 from six crystal forms that represent three distinct nucleotide -ligand binding states . One of the (Mg²⁺ )₂·ADP·GRK1 structures is the most high -resolution structure (1 .85 Å ) of a GRK to date . In one (Mg²⁺ )₂·ATP·GRK1 structure , almost the entire N -terminal region (residues 5 -30 ) is observed . In addition , different segments of the kinase C -terminal extension are ordered in the various nucleotide -bound structures . Together , these two elements form a putative receptor -docking site adjacent to the hinge of the kinase domain . Based on these structures , a model is proposed for how GRK1 interacts with activated rhodopsin and how rhodopsin binding in turn could activate the kinase . Two novel phosphorylation sites were also identified at the N -terminus . The physiological role of phosphorylation sites and the extensive dimerization interface mediated by the regulator of G protein signaling (RGS ) homology domain of GRK1 was assessed using site -directed mutagenesis . In addition to mediating interaction with activated GPCRs , the N -terminus of GRKs also forms a binding site for calcium sensing proteins . Although its physiological significance is debated , the structures of these complexes could lend further insights into the conformation of the N -terminus of GRKs . The second chapter deals with attempts to isolate Ca²⁺·recoverin·GRK1 and Ca²⁺·calmodulin·GRK6 complexes . Finally , the RH domain of GRK2 binds G[alpha subscript q] , G[alpha]₁₁ , and G[alpha]₁₄ subunits thereby blocking their interactions with the downstream effectors . The third chapter involves attempts to isolate a complex of GRK6 and G[alpha]₁₆ , a member of G[alpha subscript q] family .
URI: http : / /hdl .handle .net /2152 /3691
Date: 2008-08-29

Citation

Rhodopsin kinase structure: different nucleotide-binding states and implications for mechanism of activation of a G protein coupled receptor kinase. Doctoral dissertation, The University of Texas at Austin. Available electronically from http : / /hdl .handle .net /2152 /3691 .

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